(1) Field of the Invention
This invention relates to intracellular proteins involved in signal transduction and, more particularly, to peptides and methods for blocking signal transduction by binding to 14-3-3 proteins.
(2) Description of the Related Art
The family of proteins known as 14-3-3 proteins are expressed in a wide variety of organisms and tissues and at least seven different isoforms exist in mammalian cells (Aitken et al., Trends Biochem Sci 17:498-501, 1992; Aitken, Trends Biochem Sci 20:95-97, 1995 which are incorporated by reference). The 14-3-3 proteins appear to mediate a number of biological activities. This protein has been found to activate neuronal tyrosine and tryptophan hydroxylase (Ichimura et al., Proc Natl Acad Sci USA 85: 7084-8, 1988 which is incorporated by reference); to regulate the activity of protein kinase C (Isobe et al. FEBS Letters 308:121-124, 1991; Toker et al. Eur J Biochem 191: 421-9, 1990; Tanji et al., J. Neurochem 63: 1908-16, 1994 which are incorporated by reference); as well as to bind to and presumably regulate a number of signaling proteins including Raf-1, polyoma middle T antigen, bcr, and PI-3 kinase (Fantl et al., Nature 371: 612-4, 1994 which is incorporated by reference).
One of these proteins, Raf, constitutes a family of serine/threonine kinases involved in the transduction of signals for growth and development from the cell surface to the nucleus. Members of the Raf family of kinases include Raf-1 which is ubiquitously expressed and A-Raf and B-Raf which have restricted patterns of expression. Raf kinases are believed to be key mediators of mitogenesis and differentiation, acting through a cascade of protein kinases that is also thought to be the pathway utilized by most oncogenes in cell transformation. (See Daum et al., Trends Biochem Sci 19:474-480, 1994 which is incorporated by reference.) The activation of Raf-1 in thought to involve phosphorylation of the molecule and several phosphorylation sites have been identified. (Morrison et al., J Biol Chem 268:17309-16, 1993 which is incorporated by reference). The phosphorylated Raf protein may then bind to the 14-3-3 protein which has been suggested to be essential to activation of Raf-1 in its mediation of these events inasmuch as microinjection of 14-3-3 results in Raf-1 activation and is required for function when Raf-1 is expressed in yeast (Fantl et al., supra; Li et al., EMBO J 14: 685-96, 1995; Irie et al., Science 265:1716-1719 which are incorporated by reference). It has been suggested that binding of 14-3-3 to Raf-1 is not necessary for activation, inasmuch as another group of investigators have reported that mutant forms of Raf-1 that are unable to bind to the 14-3-3 protein nevertheless show in vitro kinase activity as well as the ability to induce meiotic maturation in oocytes thus suggesting that binding to 14-3-3 is not essential (Michaud et al., Mol Cell Biol 15:3390-3397, 1995 which is incorporated by reference). This work is based upon the idea that 14-3-3 binds to a phosphorylation site (ser-259) that is induced by growth factor treatment. But 14-3-3 is associated with Raf-1 constitutively so this phosphorylation site (ser-259) cannot be the only binding site for 14-3-3. Thus, it was not appreciated that 14-3-3 binds to a Raf-1 phosphorylation site that is essential for function as is disclosed herein.
Nevertheless, because of the role of Raf-1 and possibly also the 14-3-3 proteins in a disease process involving growth and differentiation, in particular such conditions as cancer, atherosclerosis and autoimmune disease, it would be desirable to provide a method for interrupting this signal transduction pathway and thereby provide a new approach to treating these diseases.